Journal: Glia

Article Title: Inflammatory Mediators Both Directly and Indirectly Promote Microglial Proliferation

doi: 10.1002/glia.70150

Figure Lengend Snippet: CSF2, IL3, and TNFɑ, but not other screened factors, boost microglial proliferation. (A) Schematic representation for microglial immunopanning, culture, and treatment. (B) Bar plot depicting the fold change in percentage of proliferative microglia in response to each screened factor at 10 ng/mL relative to the MGM control. (C) Representative images of CSF2, IL3, or TNFɑ induced microglial proliferation. (D) Bar plot depicting the percentage of proliferative microglia in response to 10 ng/mL CSF2. (E) Bar plot depicting the percentage of proliferative microglia in response to 10 ng/mL IL3. (F) Bar plot depicting the percentage of proliferative microglia in response to 10 ng/mL TNFɑ. (B): N = 3 independent microglial cultures with treatment in triplicate; (D–F): N = 9–12 wells across 3–4 independent microglial cultures. Bars represent mean ± SEM. B: One‐way ANOVA, Dunnett's post hoc comparing fold changes to mean MGM control; (D–F): Unpaired t ‐test; Colors represent independent microglial cultures: Large data points = mean percentage of proliferative microglia per culture, small data points = percentage of proliferative microglia per well. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar = 50 μm. BDNF, Brain‐derived neurotrophic factor; CSF, Colony stimulating factor; CX3CL1, Fractalkine; IFN, Interferon; IGF, Insulin‐like growth factor; IL, Interleukin; LPA, Lysophosphatidic acid; MGM, Microglia growth media; NGF, Nerve growth factor β; NRG1, Neuregulin 1; NT3, Neurotrophin 3; NTS, Neurotensin; S1P, Sphingosine‐1 phosphate; SCF, Stem cell factor; TNFɑ, Tumor necrosis factor ɑ; VEGF, Vascular endothelial growth factor; WNT3a, Wingless‐related int‐3a.

Article Snippet: To determine the concentration of CSF2 in our concentrated ACM, we performed a CSF2 ELISA (R&D Systems MGM00) as per the manufacturer's instructions.

Techniques: Control, Derivative Assay

Journal: Glia

Article Title: Inflammatory Mediators Both Directly and Indirectly Promote Microglial Proliferation

doi: 10.1002/glia.70150

Figure Lengend Snippet: Astrocyte‐secreted CSF2 promotes microglial proliferation in response to IL1ɑ and IL1β stimulation. (A) Plot depicting the percentage of proliferative microglia following 10 ng/mL treatment with each elevated cytokine identified by cytokine assay in microglia media lacking CSF1 and TGFβ2. (B) Plot depicting the concentration of CSF2 protein as determined by ELISA in 50× concentration ACM, IL1ɑ and IL1β ACM. (C) Representative images of 50× concentrated IL1ɑ ACM and IL1β ACM induced microglial proliferation (red = CD11b; white = KI67) and CSF2 neutralization. (D) Plot depicting the percentage of proliferative microglia in response to 50× concentrated UCM, ACM, IL1ɑ ACM with either IgG2A isotype control or ɑCSF2 treatment. (E) Plot depicting the percentage of proliferative microglia in response to 50× concentrated UCM, ACM, IL1β ACM with either IgG2A isotype control or ɑCSF2 treatment. Bars represent mean ± SEM. A: N = 12 wells from 3 independent cultures; (B): N = 4 batches of ACM; (D‐E): N = 9 wells from 3 independent cultures, the same AUCM and ACM controls were used for each of these experiments as they were prepared and tested alongside each respective IL1 ACM. (A, C, D): Colors represent independent microglial cultures: Large data points = mean percentage of proliferative microglia per culture, small data points = percentage of proliferative microglia per well. (A, B, D, E): One‐way ANOVA, Tukey's post hoc to compare differences between all conditions, * p < 0.05. Scale bars = 50 μm. ACM, Astrocyte conditioned media; CSF, Colony stimulating factor; CXCL, C‐X‐C motif chemokine ligand; CX3CL1, Fractalkine; IL, Interleukin; UCM, Unconditioned media.

Article Snippet: To determine the concentration of CSF2 in our concentrated ACM, we performed a CSF2 ELISA (R&D Systems MGM00) as per the manufacturer's instructions.

Techniques: Cytokine Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Control